Basolateral Membrane Of Hepatocytes Research Articles

Evolutionary analysis of SLC10 family members and insights into function and expression regulation of lamprey NTCP.

The Na ( +)-taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier family 10 (SLC10), which consists of 7 members (SLC10a1-SLC10a7). NTCP is a transporter localized to the basolateral membrane of hepatocytes and is primarily responsible for the absorption of bile acids. Although mammalian NTCP has been extensively studied, little is known about the lamprey NTCP (L-NTCP). Here we show that L-NTCP follows the biological evolutionary history of vertebrates, with conserved domain, motif, and similar tertiary structure to higher vertebrates. L-NTCP is localized to the cell surface of lamprey primary hepatocytes by immunofluorescence analysis. HepG2 cells overexpressing L-NTCP also showed the distribution of L-NTCP on the cell surface. The expression profile of L-NTCP showed that the expression of NTCP is highest in lamprey liver tissue. L-NTCP also has the ability to transport bile acids, consistent with its higher vertebrate orthologs. Finally, using a farnesoid X receptor (FXR) antagonist, RT-qPCR and flow cytometry results showed that L-NTCP is negatively regulated by the nuclear receptor FXR. This study is important for understanding the adaptive mechanisms of bile acid metabolism after lamprey biliary atresia based on understanding the origin, evolution, expression profile, biological function, and expression regulation of L-NTCP.

The Double-Leucine Motifs Affect Internalization, Stability, and Function of Organic Anion Transporting Polypeptide 1B1.

Organic anion transporting polypeptide 1B1 (OATP1B1) is specifically expressed at the basolateral membrane of human hepatocytes and plays important roles in the uptake of various endogenous and exogenous compounds including many drugs. The proper functioning of OATP1B1, hence, is essential for the bioavailability of various therapeutic agents and needs to be tightly regulated. Dileucine-based signals are involved in lysosomal targeting, internalization, and trans-Golgi network to endosome transporting of membrane proteins. In the current study, we analyzed the 3 intracellular and 13 transmembrane dileucine motifs (DLMs) within the sequence of OATP1B1. It was found that the simultaneous replacement of I332 and L333 with alanine resulted in a significantly reduced level of the mature form of OATP1B1. The cell surface expression of I332A/L333A could be partially rescued by MG132, as well as agents that prevent clathrin-dependent protein internalization, suggesting that this dileucine motif may be involved in the endocytosis of OATP1B1. On the other hand, I376/L377 and I642/L643, which are localized at transmembrane helices (TM) 8 and 12, respectively, are involved in the interaction of the transporter with its substrates. I642A/L643A exhibited a significantly decreased protein level compared to that of the wild-type, implying that the motif is important for maintaining the stability of OATP1B1 as well.

Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy.

Na+/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible forthe sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein-protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.

Constitutive Occurrence of E:N-cadherin Heterodimers in Adherens Junctions of Hepatocytes and Derived Tumors.

Cell–cell junctions are pivotal for embryogenesis and tissue homeostasis but also play a major role in tumorigenesis, tumor invasion, and metastasis. E-cadherin (CDH1) and N-cadherin (CDH2) are two adherens junction’s transmembrane glycoproteins with tissue-specific expression patterns in epithelial and neural/mesenchymal cells. Aberrant expression has been implicated in the process of epithelial–mesenchymal transition (EMT) in malignant tumors. We could hitherto demonstrate cis-E:N-cadherin heterodimer in endoderm-derived cells. Using immunoprecipitation in cultured cells of the line PLC as well as in human hepatocellular carcinoma (HCC)-lysates, we isolated E-N-cadherin heterodimers in a complex with the plaque proteins α- and β-catenin, plakoglobin, and vinculin. In confocal laser scanning microscopy, E-cadherin co-localized with N-cadherin at the basolateral membrane of normal hepatocytes, hepatocellular adenoma (HCA), and in most cases of HCC. In addition, we analyzed E- and N-cadherin expression via immunohistochemistry in a large cohort of 868 HCCs from 570 patients, 25 HCA, and respective non-neoplastic liver tissue, and correlated our results with multiple prognostic markers. While E- or N-cadherin were similarly expressed in tumor sites with vascular invasion or HCC metastases, HCC with vascular encapsulated tumor clusters (VETC) displayed slightly reduced E-cadherin, and slightly increased N-cadherin expression. Analyzing The Cancer Genome Atlas patient cohort, we found that reduced mRNA levels of CDH1, but not CDH2 were significantly associated with unfavorable prognosis; however, in multivariate analysis, CDH1 did not correlate with prognosis. In summary, E- and N-cadherin are specific markers for hepatocytes and derived HCA and HCC. E:N-cadherin heterodimers are constitutively expressed in the hepatocytic lineage and only slightly altered in malignant progression, thereby not complying with the concept of EMT.

How does S‐palmitoylation affect the Organic Anion Transporting Polypeptide 1B1 (OATP1B1)?

BackgroundOrganic Anion Transporting Polypeptide 1B1 (OATP1B1) is an important transporter localized to the basolateral membrane of human hepatocytes that mediates the uptake of numerous therapeutics like statins, antibiotics, and anti‐cancer agents. Previous studies from our lab suggest that OATP1B1 is localized in membrane microdomains, also known as lipid rafts, which contain various components like saturated lipids, cholesterol, and lipidated proteins. Lipidated proteins are frequently the result of S‐palmitoylation, a reversible post translational modification that adds palmitic acid (C16:0) to cysteine residues. Currently, not much is known about the potential impact of S‐palmitoylation on OATP1B1 function and localization. Aim: We hypothesize that OATP1B1 is S‐palmitoylated and that this affects its function and localization.MethodsWe measured uptake of the model substrate estradiol‐17β‐glucuronide in cells expressing OATP1B1. S‐palmitoylation was detected using an acyl‐biotin exchange (ABE) assay in combination with western blotting. To prevent S‐palmitoylation, cells were preincubated with 2‐bromopalmitate (2BP). Expression of OATP1B1 at the plasma membrane was assessed using surface biotinylation.ResultsThe ABE assay demonstrated that OATP1B1 is S‐palmitoylated and that pretreatment of the cells for 18 hours with 2BP reduces S‐palmitoylation. Inhibition of S‐palmitoylation with 2BP for 18 hours also resulted in decreased OATP1B1‐mediated transport of estradiol‐17β‐glucuronide as well as in a reduction of surface expression of OATP1B1.Summary and ConclusionsOur results demonstrate that OATP1B1 is S‐palmitoylated. Inhibition of S‐palmitoylation reduces the function and the surface expression of OATP1B1. Thus, these findings suggest that S‐palmitoylation is a post‐translational modification involved in the regulation of this important hepatic drug uptake transporter.

Aquaporin-9 facilitates liver regeneration following hepatectomy

Aquaporin-9 (AQP9) is an aquaglyceroporin strongly expressed in the basolateral membrane of hepatocytes facing the sinusoids. AQP9 is permeable to hydrogen peroxide (H2O2) and glycerol as well as to water. Here, we report impaired liver regeneration in AQP9−/− mice which involves altered steady-state H2O2 concentration and glucose metabolism in hepatocytes. AQP9−/− mice showed remarkably delayed liver regeneration and increased mortality following 70% or 90% partial hepatectomy. Compared to AQP9+/+ littermates, AQP9−/− mice showed significantly greater hepatic H2O2 concentration and more severe liver injury. Fluorescence measurements indicated impaired H2O2 transport across plasma membrane of primary cultured hepatocytes from AQP9−/− mice, supporting the hypothesis that AQP9 deficiency results in H2O2 accumulation and oxidative injury in regenerating liver because of reduced export of intracellular H2O2 from hepatocytes. The H2O2 overload in AQP9−/− hepatocytes reduced PI3K-Akt and insulin signaling, inhibited autophagy and promoted apoptosis, resulting in impaired proliferation and increased cell death. In addition, hepatocytes from AQP9−/− mice had low liver glycerol and high blood glycerol levels, suggesting decreased glycerol uptake and gluconeogenesis in AQP9−/− hepatocytes. Adeno-associated virus (AAV)-mediated expression of hepatic expression of aquaglyceroporins AQP9 and AQP3 in AQP9−/− mice, but not water-selective channel AQP4, fully rescued the impaired liver regeneration phenotype as well as the oxidative injury and abnormal glucose metabolism. Our data revealed a pivotal role of AQP9 in liver regeneration by regulating hepatocyte H2O2 homeostasis and glucose metabolism, suggesting AQP9 as a novel target to enhance liver regeneration following injury, surgical resection or transplantation.

Effect of major components of Tripterygium wilfordii Hook. f on the uptake function of organic anion transporting polypeptide 1B1

Organic anion transporting polypeptide 1B1 (OATP1B1), which is specifically expressed at the basolateral membrane of human hepatocytes, is well recognized as the key determinant in the pharmacokinetics of a wide variety of drugs and considered as an important drug-drug interaction (DDI) site. Triptergium wilfordii Hook. f. (TWHF) is a traditional Chinese medicine that has a long history in treating diseases and more pharmacological effects were demonstrated recently. Components of TWHF mainly belong to the groups of alkaloids, diterpenoids, and triterpenoids. However, whether TWHF constituents are involved in herb-drug interaction (HDI) remains largely unknown. In the present study, we investigated the effect of four major components of TWHF, i.e. Triptolide (TPL), Celastrol (CL), and two alkaloids Wilforine (WFR) and Wilforgine (WFG) on the function of OATP1B1. It was found that co-incubation of these compounds greatly inhibited the uptake function of OATP1B1, with WFG (IC50 = 3.63 ± 0.61 μM) and WFR (IC50 = 3.91 ± 0.30 μM) showing higher inhibitory potency than TPL (IC50 = 184 ± 36 μM) and CL (IC50 = 448 ± 81 μM). Kinetic analysis revealed that co-incubation of WFG or WFR led to the reduction of both Km and Vmax of the DCF uptake. On the other hand, pre-incubation of WFG or WFR increased Km value of OATP1B1; while CL affected both Km and Vmax. In conclusion, co- and pre-incubation of the tested TWHF components inhibited OATP1B1 activity in different manners. Although co-incubation of WFG and WFR did not seem to directly compete with the substrates, pre-incubation of these alkaloids may alter the substrate-transporter interaction.

Human Organic Anion Transporting Polypeptides 1B1, 1B3, and 2B1 Are Involved in the Hepatic Uptake of Phenolsulfonphthalein.

Phenolsulfonphthalein (PSP or phenol red), a sulfonphthalein dye, has been used as a diagnostic agent and a pH indicator in cell culture medium. After administered into the body, PSP is excreted into urine and bile. The urinary excretion of PSP is mediated by organic anion transporter 1/3 (OAT1/3) and multidrug resistance protein 2 (MRP2). In biliary excretion, PSP is effluxed from hepatocytes into the bile via MRP2. However, so far, the molecular mechanism for PSP transport from the blood into hepatocytes is unclear. In the present study, six human major hepatic uptake transporters expressed on the basolateral membrane of hepatocytes, namely, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, Na+/taurocholate cotransporting polypeptide (NTCP), organic cation transporter 1 (OCT1), and OAT2, have been investigated to see whether they are involved in the hepatic uptake of PSP. An in vitro cell-based study demonstrated that PSP is a substrate for OATP1B1, OATP1B3, and OATP2B1, with OATP1B3 showing the highest transport efficiency. The Km values for OATP1B1-, OATP1B3-, and OATP2B1-mediated PSP uptake were 11.3 ± 1.5, 7.0 ± 1.5, and 5.1 ± 1.0 μM, respectively. PSP interacts with known OATP substrates/inhibitors. However, the presence of PSP in cell culture medium has no significant effect on OATP’s function. In vivo pharmacokinetic study in wild-type and Oatp1b2-knockout mice showed that Oatp1b2-knockout led to elevated plasma concentration and decreased liver accumulation of PSP. Taken together, the present study showed that in the liver, OATP1B1, OATP1B3, and OATP2B1 are involved in the uptake of PSP from the blood into hepatocytes, which, along with MRP2-mediated efflux of PSP from hepatocytes into the bile, constitute the vectorial transport of PSP from the blood to the bile and may play a critical role in the biliary excretion of PSP.

Pre-incubation with OATP1B1 and OATP1B3 inhibitors potentiates inhibitory effects in physiologically relevant sandwich-cultured primary human hepatocytes

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 are liver-specific transport proteins that express on the basolateral membrane of human hepatocytes and mediate hepatic uptake of many drugs, including statins. They are important determinants of transporter-mediated drug-drug interactions (DDIs). It has been reported that pre-incubation with some OATP1B1 and OATP1B3 inhibitors potentiates the inhibitory effects, yielding reduced IC50 values. The US FDA draft guidance has recently recommended to use the lower IC50 values after inhibitor-preincubation to assess OATP1B1 and OATP1B3-mediated DDIs. However, it remains unknown whether the potentiation effects of inhibitor-preincubation on IC50 values occur in a physiologically relevant cell model. The current study was designed to determine the IC50 values of OATP1B1 and OATP1B3 inhibitors everolimus (EVR), sirolimus (SIR), and dasatinib against OATP1B substrates in physiologically relevant primary human hepatocytes with or without inhibitor-preincubation and to compare the OATP-mediated DDI prediction using data from primary human hepatocytes and that reported previously in transporter-expressing cell lines. Primary human hepatocytes were cultured in a sandwich configuration. Accumulation of [3H]-CCK-8 (1 µM, 1.5 min), [3H]-rosuvastatin (0.5 µM, 2 min) and [3H]-pitavastatin (1 µM, 0.5 min) was determined in human sandwich-cultured hepatocytes (SCH) in the presence of vehicle control or an inhibitor with or without inhibitor-preincubation at designated concentrations, and was utilized to determine the IC50 values for these inhibitors. R-value models were used to predict OATP-mediated DDIs. Pre-incubation with EVR at a clinically relevant concentration of 0.2 µM significantly reduced accumulation of [3H]-CCK-8 and [3H]-rosuvastatin even after washing. Reduced IC50 values following inhibitor pre-incubation were observed for all three inhibitors using [3H]-CCK-8 and [3H]-rosuvastatin as substrates in human SCH. The IC50 values after inhibitor-preincubation were lower or comparable in transporter-expressing cell lines compared with that in human SCH. For dasatinib, R-values from both cell lines and human SCH were greater than the US FDA cut-off value of 1.1. For EVR, R values from cell lines were 1.23 and were lowered to near 1.1 (1.08–1.09) in human SCH. For SIR, R values from either cell type were less than the cut-off values of 1.1. In conclusion, the current study is the first to report that pre-incubation with OATP1B inhibitors potentiates inhibitory effects in physiologically relevant primary human hepatocytes, supporting the rationale of the current US FDA draft guidance of including an inhibitor-preincubation step when assessing OATP-mediated DDIs in vitro. IC50 values after inhibitor-preincubation in transporter-expressing cell lines may be used for DDI prediction for the purpose of mitigating false-negative OATP-mediated DDI prediction.

Imaging-Based Characterization of a Slco2b1(-/-) Mouse Model Using [11C]Erlotinib and [99mTc]Mebrofenin as Probe Substrates.

Organic anion-transporting polypeptide 2B1 (OATP2B1) is co-localized with OATP1B1 and OATP1B3 in the basolateral hepatocyte membrane, where it is thought to contribute to the hepatic uptake of drugs. We characterized a novel Slco2b1(-/-) mouse model using positron emission tomography (PET) imaging with [11C]erlotinib (a putative OATP2B1-selective substrate) and planar scintigraphic imaging with [99mTc]mebrofenin (an OATP1B1/1B3 substrate, which is not transported by OATP2B1). Dynamic 40-min scans were performed after intravenous injection of either [11C]erlotinib or [99mTc]mebrofenin in wild-type and Slco2b1(-/-) mice. A pharmacokinetic model was used to estimate the hepatic uptake clearance (CL1) and the rate constants for transfer of radioactivity from the liver to the blood (k2) and excreted bile (k3). CL1 was significantly reduced in Slco2b1(-/-) mice for both radiotracers (p < 0.05), and k2 was significantly lower (p < 0.01) in Slco2b1(-/-) mice for [11C]erlotinib, but not for [99mTc]mebrofenin. Our data support previous evidence that OATP transporters may contribute to the hepatic uptake of [11C]erlotinib. However, the decreased hepatic uptake of the OATP1B1/1B3 substrate [99mTc]mebrofenin in Slco2b1(-/-) mice questions the utility of this mouse model to assess the relative contribution of OATP2B1 to the liver uptake of drugs which are substrates of multiple OATPs.

Data-Driven Ensemble Docking to Map Molecular Interactions of Steroid Analogs with Hepatic Organic Anion Transporting Polypeptides.

Hepatic organic anion transporting polypeptides—OATP1B1, OATP1B3, and OATP2B1—are expressed at the basolateral membrane of hepatocytes, being responsible for the uptake of a wide range of natural substrates and structurally unrelated pharmaceuticals. Impaired function of hepatic OATPs has been linked to clinically relevant drug–drug interactions leading to altered pharmacokinetics of administered drugs. Therefore, understanding the commonalities and differences across the three transporters represents useful knowledge to guide the drug discovery process at an early stage. Unfortunately, such efforts remain challenging because of the lack of experimentally resolved protein structures for any member of the OATP family. In this study, we established a rigorous computational protocol to generate and validate structural models for hepatic OATPs. The multistep procedure is based on the systematic exploration of available protein structures with shared protein folding using normal-mode analysis, the calculation of multiple template backbones from elastic network models, the utilization of multiple template conformations to generate OATP structural models with various degrees of conformational flexibility, and the prioritization of models on the basis of enrichment docking. We employed the resulting OATP models of OATP1B1, OATP1B3, and OATP2B1 to elucidate binding modes of steroid analogs in the three transporters. Steroid conjugates have been recognized as endogenous substrates of these transporters. Thus, investigating this data set delivers insights into mechanisms of substrate recognition. In silico predictions were complemented with in vitro studies measuring the bioactivity of a compound set on OATP expressing cell lines. Important structural determinants conferring shared and distinct binding patterns of steroid analogs in the three transporters have been identified. Overall, this comparative study provides novel insights into hepatic OATP-ligand interactions and selectivity. Furthermore, the integrative computational workflow for structure-based modeling can be leveraged for other pharmaceutical targets of interest.

Functional and Pharmacological Comparison of Human and Mouse Na+/Taurocholate Cotransporting Polypeptide (NTCP).

The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.

Transport of Drugs and Endogenous Compounds Mediated by Human OCT1: Studies in Single- and Double-Transfected Cell Models.

Organic Cation Transporter 1 (OCT1, gene symbol: SLC22A1) is predominately expressed in human liver, localized in the basolateral membrane of hepatocytes and facilitates the uptake of endogenous compounds (e.g. serotonin, acetylcholine, thiamine), and widely prescribed drugs (e.g. metformin, fenoterol, morphine). Furthermore, exogenous compounds such as MPP+, ASP+ and Tetraethylammonium can be used as prototypic substrates to study the OCT1-mediated transport in vitro. Single-transfected cell lines recombinantly overexpressing OCT1 (e.g., HEK-OCT1) were established to study OCT1-mediated uptake and to evaluate transporter-mediated drug-drug interactions in vitro. Furthermore, double-transfected cell models simultaneously overexpressing basolaterally localized OCT1 together with an apically localized export protein have been established. Most of these cell models are based on polarized grown MDCK cells and can be used to analyze transcellular transport, mimicking the transport processes e.g. during the hepatobiliary elimination of drugs. Multidrug and toxin extrusion protein 1 (MATE1, gene symbol: SLC47A1) and the ATP-driven efflux pump P-glycoprotein (P-gp, gene symbol: ABCB1) are both expressed in the canalicular membrane of human hepatocytes and are described as transporters of organic cations. OCT1 and MATE1 have an overlapping substrate spectrum, indicating an important interplay of both transport proteins during the hepatobiliary elimination of drugs. Due to the important role of OCT1 for the transport of endogenous compounds and drugs, in vitro cell systems are important for the determination of the substrate spectrum of OCT1, the understanding of the molecular mechanisms of polarized transport, and the investigation of potential drug-drug interactions. Therefore, the aim of this review article is to summarize the current knowledge on cell systems recombinantly overexpressing human OCT1.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

Effect of bile acids on the expression of MRP3 and MRP4: An In vitro study in HepG2 cell line

Introduction and objectivesFree and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters’ increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters’ mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. MethodsThe expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. ResultsThe results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. ConclusionPrimary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.

OATP1B3 Expression and Function is Modulated by Coexpression with OCT1, OATP1B1, and NTCP.

Organic anion transporting polypeptide (OATP) 1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes. Along with other transporters, including OATP1B1, Na+/taurocholate cotransporting polypeptide (NTCP), and organic cation transporter (OCT) 1, it is responsible for the uptake of endo- and xenobiotics into hepatocytes. Our previous studies demonstrated that OATP1B3 can form hetero-oligomers with OATP1B1 in human embryonic kidney 293T (HEK293) cells and with NTCP in both HEK293 cells and frozen human liver sections. To further characterize the hetero-oligomerization of OATP1B3, we investigated OCT1 as a potential interacting partner and determined the functional consequences of OATP1B3 hetero-oligomerization. We demonstrated interactions between OATP1B3 and OCT1 by coimmunoprecipitation with an anti-OATP1B3 antibody from human hepatocytes. In addition, we visualized the interaction using the proximity ligation assay in both HEK293 cells and in frozen human liver sections. We investigated the functional consequences of OATP1B3 hetero-oligomerization by measuring the OATP1B3 plasma membrane expression and the uptake of the OATP1B3 selective substrate cholecystokinin-8 (CCK-8) in the absence and presence of OATP1B1, NTCP, and OCT1. A significant decrease of OATP1B3 plasma membrane expression was observed after coexpression with OCT1, whereas coexpression with OATP1B1 or NTCP resulted in an increase of plasma membrane expression. With respect to transport, coexpression of OCT1 increased the apparent turnover rate of OATP1B3, whereas coexpression of OATP1B1 or NTCP decreased it. These findings demonstrated that coexpression of OATP1B3 with OATP1B1, NTCP, and OCT1 in HEK293 cells results in a transporter-dependent modification of OATP1B3-mediated CCK-8 transport and suggest that functional results obtained in single transporter overexpressing cell lines over- or underestimate OATP1B3 function in human hepatocytes. SIGNIFICANCE STATEMENT: Coexpression of organic anion transporting polypeptide (OATP) 1B3 with organic cation transporter (OCT) 1, Na+/taurocholate cotransporting polypeptide, or OATP1B1 in human embryonic kidney 293T cells affects its expression level and function. When OCT1 is knocked down in human hepatocytes, function of OATP1B3 goes up. These results suggest that protein-protein interactions can affect the expression and function of the involved proteins, and thus single transporter expression systems might lead to over- or underestimation of drug-drug interactions.

The Lipid Raft Component Stomatin Interacts with the Na+ Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake.

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein–protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.

The Function of Hepatocellular Uptake Transporters is Affected by Free Cholesterol

BackgroundWe have previously shown that several transporters located in the basolateral membrane of human hepatocytes e.g. NTCP, OCT1, OATP1B1, and OATP1B3 interact with each other. These interactions are due in part to their co‐localization in detergent resistant membrane domains and impact both function and expression. Understanding how transporter interaction and altered membrane cholesterol content impact the function of these drug uptake transporters has implications regarding pharmacological studies, drug development, and drug‐drug interaction predictions.AimWe wanted to test how both the addition and removal of free cholesterol to and from the plasma membrane alters the function and localization of drug transporters.MethodsThe function of NTCP or OCT1 was measured using HEK293 Flp‐In cells stably expressing either transporter. We treated the cells with methyl‐b‐cyclodextrin (MCD) to remove cholesterol or with a cholesterol‐MCD complex to add cholesterol. Lipid rafts were isolated from HEK293 Flp‐In cells or frozen human liver samples by homogenization in 1% Triton X‐100 and sucrose gradient centrifugation. Gradient fractions were analyzed by western blotting using positive and negative lipid raft markers as well as transporter specific antibodies.ResultsTreatment with MCD stimulated uptake by NTCP while inhibiting uptake by OCT1. Subsequent treatment of the MCD treated cells with the cholesterol‐MCD complex restored function in part for both NTCP and OCT1. Removal and restoration of membrane free cholesterol also impacted the co‐localization of the transporters within the detergent resistant membrane domains. We confirmed the differential localization of transporters in detergent resistant membranes comparing samples of normal and fatty human livers.Summary and ConclusionBoth adding or removing cholesterol alters transport function in a transporter‐dependent way. Furthermore, the localization of transporters in lipid rafts is different in normal and in fatty human livers. These results suggest that drug disposition is affected by fatty liver disease and that some drugs may be cleared faster or slower in patients with increased free cholesterol in the liver, due to NAFLD or NASH.Support or Funding InformationR01GM077336 and P30GM118247

Functional Characterization of Position 271 in NTCP, a Predicted Rheostat Location

BackgroundHuman Na+/taurocholate cotransporting polypeptide (NTCP) is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. It is important for the enterohepatic circulation of bile acids, such as taurocholate (TCA), but it also transports other compounds including estrone‐3‐sulfate (E3S) and rosuvastatin. We previously identified a rheostat position at a naturally occurring polymorphism, S267F. That is, when other amino acids were substituted into this position, progressive changes in function were observed.AimThe purpose of the current study is to determine whether position N271, a predicted rheostat, is indeed another rheostat position in NTCP.MethodsThe crystal structures of two bacterial NTCP homologs were used to model the structure of human NTCP in the inward‐open and outward‐open conformations. Mutations were then introduced into the structural models and the energetic effect on protein stability was evaluated using the Rosetta software. Based on these calculations we predicted that N271 is a rheostat. Mutants for position N271 were generated using site‐directed mutagenesis and the resulting proteins were transiently expressed in HEK293 cells. The functions of the mutants were characterized by measuring uptake of radiolabeled model substrates: [3H]‐taurocholate, [3H]‐estrone‐3‐sulfate, and [3H]‐rosuvastatin. Plasma membrane expression levels were determined by surface biotinylation assays.ResultsInitial transport studies using nanomolar concentrations of TCA, E3S, and rosuvastatin indicated a variety of intermediate functional outcomes for the mutants. Furthermore, this alteration in function was substrate dependent, which is consistent with our findings for S267. Some mutants, such as N271A, transport substrates similar to wild‐type NTCP, while others, like N271K, show variation depending on the substrate. N271P consistently transports all substrates minimally. In addition, there is no correlation between the initial uptake results and the Rosetta energies.Summary and ConclusionsInitial transport studies demonstrate that position N271 is a rheostat position, as predicted by the alteration in Rosetta energy scores. However, there was no correlation between the calculated energy scores and the functional results. This suggests that functional outcomes of specific amino acid replacements at position N271 may arise from more complex conformational changes than were captured by the two modeled states. Additional experiments should help to determine to what extent the observed change in function is due to variations in the substrate affinity, the turnover number or the transport capacity of the mutants.Support or Funding InformationP30 GM118247, GM077336, and W. M. Keck Foundation

Role of Oatp2b1 in Drug Absorption and Drug-Drug Interactions.

The organic anion transporting polypeptide (OATP)2B1 is localized on the basolateral membrane of hepatocytes and is expressed in enterocytes. Based on its distribution pattern and functional similarity to OATP1B-type transporters, OATP2B1 might have a role in the absorption and disposition of a range of xenobiotics. Although several prescription drugs, including hydroxymethylglutaryl-coenzyme A-CoA reductase inhibitors (statins) such as fluvastatin, are OATP2B1 substrates in vitro, evidence supporting the in vivo relevance of this transporter remains limited, and most has relied on substrate-inhibitor interactions resulting in altered pharmacokinetic properties of the victim drugs. To address this knowledge deficit, we developed and characterized an Oatp2b1-deficient mouse model and evaluated the impact of this transporter on the absorption and disposition of fluvastatin. Consistent with the intestinal localization of Oatp2b1, we found that the genetic deletion or pharmacological inhibition of Oatp2b1 was associated with decreased absorption of fluvastatin by 2- to 3-fold. The availability of a viable Oatp2b1-deficient mouse model provides an opportunity to unequivocally determine the contribution of this transporter to the absorption and drug-drug interaction potential of drugs. SIGNIFICANCE STATEMENT: The current investigation suggests that mice deficient in Oatp2b1 provide a valuable tool to study the in vivo importance of this transporter. In addition, our studies have identified novel potent inhibitors of OATP2B1 among the class of tyrosine kinase inhibitors, a rapidly expanding class of drugs used in various therapeutic areas that may cause drug-drug interactions with OATP2B1 substrates.